The ability to differentiate and phenotype blood cells is useful for evaluating disease states and other health conditions in living beings. One popular technique for cell differentiation and lymphocyte immunophenotyping is flow cytometry. With flow cytometry, cells from an appropriately prepared blood sample, are passed one at a time through a flow cell, which is adapted for sensing or detecting impedance changes, light scatter or some other characteristic of the cell. Some flow cytometry instruments are equipped with detectors for measuring emissions from fluorescent tags that may be associated with the cells, while other detectors measure scatter intensity or pulse duration. Data about cells that pass through the flow cell can be plotted on a cytogram according to the measured property.
During the flow cytometry process, it is important that interference from the presence of erythrocytes (red blood cells) in the blood sample be avoided. Accordingly, during sample preparation, which may be done by manual, semi-automated or automated techniques), it is popular to employ a lytic reagent for lysing red blood cells and thereby isolating the leukocyte (white blood cell) populations. Leukocytes are known to include a myeloid fraction of monocytes and granulocytes (neutrophils, basophils and eosinophils) and a lymphoid fraction (namely NK, B and T cell lymphocytes). Each of the lymphocyte populations can be distinguished based upon the distinctive cell surface antigens or markers. Moreover, within each category of lymphocytes, there are sub-categories, such as “helper” T cells or “suppressor” T cells, the latter of which also includes several subsets, distinguishable by their respective surface markers. With flow cytometry of properly prepared cells, using polyclonal or monoclonal antibodies, it is possible to assay lymphocytes to analyze cells in the various subcategories.
For instance, to prepare a sample for fluorescent flow cytometry, according to one conventional practice, a volume of fresh sample blood is provided, and a suitable amount of a desired fluorochrome labeled antibody is added. The sample and antibody mixture is incubated to allow antibody/antigen bindings to take place. After incubation, a lytic reagent (some of which are regarded as potentially toxic, i.e., those containing formaldehyde) is added to lyse erythrocytes in the sample. The debris from the lysing of the erythrocytes is optionally removed, by washing, leaving a sample of leukocytes with antibodies bound to cells with complementary surface antigens. The sample is fixed and run through a fluorescent detecting flow cytometry instrument.
Among the items of potential interest to the present invention are Brown et al, “Flow Cytometry: Principles and Clinical Applications in Hematology”, Clinical Chemistry 46:8(B), 1221–1229 (2000); U.S. Pat. Nos. 4,654,312; 4,902,613; 5,030,554; 5,188,935; 5,196,182; 5,250,438; 5,260,048; 5,459,073; 5,460,797; 5,731,206; 5,776,709; 5,811,099; 5,849,517, 5,939,326; 6,110,730; and commonly owned, co-pending U.S. patent application Ser. No. 09/500,248 (“Fixative System, Method and Composition for Biological Testing”), the teachings of each of which are hereby expressly incorporated by reference for all purposes.
Accordingly, in view of the above, there is a need in the art for an improved lytic reagent that lyses red blood cells systematically and reproducibly preserves the surface antigen characteristics of leukocytes; that enables fixing of the leukocytes during the erythrocyte lysing step; that is nontoxic; and that can be used to prepare samples for analysis beyond one day.